In the brain, normal and pathological electrical activity gives rise to rapid changes in extracellular and intracellular pH. This modulation of pH can feedback to influence neural activity and can impact the response to brain hypoxia and ischemia. It is known that the intracellular pH of glial cells increases in response to membrane depolarization, due to sodium driven, electrogenic, entry of bicarbonate. This influx simultaneously acts to acidify the extracellular fluid. By contrast, depolarization of neurons slowly acidifies the cytosol, a response associated with entry of calcium. Preliminary studies on hippocampal neurons indicate that such neural responses represent a balance between a calcium dependent acidification, and a nearly equivalent alkalinizing mechanism that is triggered by depolarization. These results suggest that neurons regulate their pH preemptively, utilizing one or more transporters responsive to membrane potential. Unlike the transport mechanism of glia, preliminary data indicate that neurons alkalinize in response to depolarization by one mechanism that requires chloride ions, and a separate process that is chloride-independent. Elucidation of these two mechanisms occupies the first two aims of this proposal. The third aim seeks to clarify their functional relevance in response to acidosis, repetitive firing, and hypoxic-ischemic insults. The last two aims concern the role of neurons in rapid regulation of extracellular pH. The fourth aim focuses on an extracellular carbonic anhydrase (type 14) localized to neurons in the hippocampus, and posits that this enzyme regulates pH in the fluid around synapses. The last aim focuses again on role of chloride dependent bicarbonate transport, but from the extracellular perspective. The project will employ tissue from mice with gene deletions of specific bicarbonate transporters. Studies will be conducted with intracellular pH imaging and whole cell recording techniques, complemented by quantitative polymerase chain reaction protocols to quantify transporter expression. Results of these experiments offer potentially groundbreaking insights into how pH is regulated in the CNS, and now that regulation impacts normal physiology, and the response to conditions such as hypoxia, cardiac arrest, stroke and traumatic brain injury. PUBLIC HEALTH RELEVANCE: This project will focus on the mechanisms used by nerve cells to regulate internal (cytoplasmic) acid base balance, during both normal and abnormal electrical activity. Elucidation of these mechanisms is critical to understanding how the brain maintains an internal microenvironment conducive to proper function. Moreover, this information should provide important new insights into how nerve cells respond to conditions such as cardiac arrest, stroke and traumatic brain injury.